Project Summary Depending on the signals in vitro, macrophages can undergo transient and reversible differentiation into two main subsets: pro-inflammatory ?M1? (classically activated) or anti-inflammatory ?M2? (alternatively activated) phenotype. Upon exposure of macrophages to high levels of glucose in vitro or in diabetic patients in vivo, they undergo differentiation and activation into a M1 pro-inflammatory state, which produces pro-inflammatory cytokines mediated by cellular reprogramming of metabolism through upregulating aerobic glycolysis. The M1 pro-inflammatory differentiation of macrophages is an import arm of the innate immune response to control bacterial infections. Our preliminary data show that when macrophages engulf the intracellular bacterial pathogen Legionella pneumophila (Lp), they rapidly respond by differentiation into an activated M1-like pro-inflammatory phenotype. Despite this rapid macrophage pro-inflammatory activation, the Legionella-containing vacuole (LCV) bypasses macrophage activation by limiting its fusion to lysosomes. We show that the early M1-like pro-inflammatory differentiation of human monocytes-derived macrophages (hMDMs) to Lp is an innate immune response to cytosolic hyper-glucose generated through rapid degradation of glycogen by the injected Legionella amylase (LamA) effector. We discovered that Lp has evolved a lysosomal degradation bypass pathway within the pro-inflammatory hMDMs by injecting the MavE effector, which becomes localized in a micro-domain on the cytosolic side of the LCV membrane. The mavE-deficient mutant is targeted by hMDMs to the lysosomes for degradation, and the mutant is totally attenuated in vitro and in vivo. We have resolved the novel three dimensional crystal structure of MavE, which shows a cytosolic surface-exposed domain with an NPxY eukaryotic motif involved in binding to phosphotyrosine-binding adaptor proteins. Our central hypothesis is: the M1-like pro- inflammatory activation of hMDMs to Lp infection is an innate immune response to the cytosolic hyper-glucose elicited by LamA, and the pathogen utilizes MavE to bypass the innate immune macrophage pathway of lysosomal degradation. To test the hypothesis, our specific aims are: Specific Aim I: The mechanisms of M1-like pro-inflammatory differentiation of hMDMs in response to the effect of LamA; and Specific Aim II: The mechanisms of evasion of the innate macrophage function of lysosomal degradation through utilization of a lysosomes bypass pathway mediated by MavE. Upon completion of our proposed studies, we will discover the mechanism of the innate pro-inflammatory response of macrophages to Lp and the lysosomal degradation bypass pathway maneuvered by the pathogen to avoid a fatal fate.